Netrin-1 is a pleiotropic peptide signaling molecule. Its most well-known role in vertebrate development is neuronal guidance. Depending upon the cell type and signal concentration gradient, netrin-1 may serve either as a chemoattractant, causing formation of axonal growth cones, or as a chemorepellent, causing growth cone collapse within the axon.
Netrin-1 can bind to at least two types of receptors, and uses a variety of signaling proteins to convey its message. In some vertebrate cell types, the netrin-1 signal is G-protein mediated, while in other cell types, netrin signaling requires a tyrosine kinase or some other combination of kinases in order to signal.
Tetrahymena thermophila are free-living, eukaryotic cells that can respond to chemoattractants and chemorepellents by moving toward attractants and away from repellents. By studying the behavior of these organisms, we have found that netrin-1 acts as a chemorepellent in T. thermophila. Response to netrin-1 is concentration dependent, with an EC100 of approximately 1 micromolar, and an EC50 of approximately 10 pM.
Netrin-1 avoidance may be effectively eliminated by the addition of the broad-spectrum tyrosine kinase inhibitor, genistein, to the behavioral assay. The IC100 of genistein was approximately 75 micrograms/ml, while the IC50 of this compound was near 50 micrograms/ml. G-protein inhibitors, calcium chelators, and a number of other pharmacological inhibitors had no effect on netrin-1 signaling in this organism.
These data show that netrin-1 is a chemorepellent in Tetrahymena thermophila and that netrin signaling appears to implicate a tyrosine kinase in this organism. Further studies will help us to determine whether genistein is specifically acting upon a tyrosine kinase pathway or whether the inhibition is occurring via some other genistein-mediated effect
